title: Measuring carbonic anhydrase IX as a hypoxia biomarker: differences in concentrations in serum and plasma using a commercial enzyme-linked immunosorbent assay due to influences of metal ions.
abstract: BACKGROUND
  There is increasing interest in measuring the soluble forms of carbonic anhydrase IX (CA IX) in blood as a marker of hypoxia for prognostic purposes or for predictive use in therapeutic trials in various cancers. Following our initial observations of marked differences in the measured concentrations of CA IX in EDTA plasma versus serum, we sought to investigate these further in order to determine their effects on results in published studies and to ensure accurate measurement in future studies.
  
  
  METHODS
  Serum and EDTA plasma samples from healthy controls and patients with renal cancer were used in the validation of two commercially available enzyme-linked immunosorbent assays (ELISAs) for CA IX with examination of recovery, parallelism and specificity and comparison of paired plasma and serum.
  
  
  RESULTS
  Successful validation of one of the ELISAs was not achieved with particular problems with parallelism and marked differences in measured CA IX concentrations between EDTA plasma and serum. This appeared to be due to a metal ion-dependent epitope on CA IX recognized by the detection antibody in this assay. The other commercially available ELISA examined was successfully validated and showed no difference in CA IX between EDTA plasma and serum.
  
  
  CONCLUSIONS
  These results have important consequences for published studies using this assay where the conclusions drawn from the measurements made may be invalid. This study highlights the need for stringent validation of commercially available assays, including examination of various sample types, before use in research studies.
authors.affiliation: Clinical and Biomedical Proteomics Group, Cancer Research UK Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK.
authors.forename: Tobias C
authors.initials: TC
authors.lastname: Wind
authors.forename: Michael P
authors.initials: MP
authors.lastname: Messenger
authors.forename: Douglas
authors.initials: D
authors.lastname: Thompson
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Selby
authors.forename: Rosamonde E
authors.initials: RE
authors.lastname: Banks
mesh_terms: D000328:Adult; D000368:Aged; D000951:Antigens, Neoplasm / Q000097:blood*; D016477:Artifacts; D015415:Biomarkers / Q000097:blood; D001774:Blood Chemical Analysis / Q000379:methods*; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000097:blood*; D004492:Edetic Acid / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D000860:Hypoxia / Q000097:blood*; D008297:Male; D008670:Metals; D008875:Middle Aged; D010949:Plasma / Q000737:chemistry*; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry*; D055815:Young Adult
chemical_list: D000951:Antigens, Neoplasm; D015415:Biomarkers; D008670:Metals; D004492:Edetic Acid; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases